An expanded biological repertoire for Ins(3,4,5,6)P4 through its modulation of ClC-3 function.
Ins(3,4,5,6)P(4) inhibits plasma membrane Cl(-) flux in secretory epithelia [1]. However, in most other mammalian cells, receptor-dependent elevation of Ins(3,4,5,6)P(4) levels is an "orphan" response that lacks biological significance [2]. We set out to identify Cl(-) channel(s) and/or transporter(s) that are regulated by Ins(3,4,5,6)P4 in vivo. Several candidates [3-5] were excluded through biophysical criteria, electrophysiological analysis, and confocal immunofluorescence microscopy. Then, we heterologously expressed ClC-3 in the plasma membrane of HEK293-tsA201 cells; whole-cell patch-clamp analysis showed Ins(3,4,5,6)P4 to inhibit Cl(-) conductance through ClC-3. Next, we heterologously expressed ClC-3 in the early endosomal compartment of BHK cells; by fluorescence ratio imaging of endocytosed FITC-transferrin, we recorded intra-endosomal pH, an in situ biosensor for Cl(-) flux across endosomal membranes [6]. A cell-permeant, bioactivatable Ins(3,4,5,6)P4 analog elevated endosomal pH from 6.1 to 6.6, reflecting inhibition of ClC-3. Finally, Ins(3,4,5,6)P(4) inhibited endogenous ClC-3 conductance in postsynaptic membranes of neonatal hippocampal neurones. Among other ClC-3 functions that could be regulated by Ins(3,4,5,6)P4 are tumor cell migration [7], apoptosis [8], and inflammatory responses [9]. Ins(3,4,5,6)P4 is a ubiquitous cellular signal with diverse biological actions.
Mitchell
Jennifer
J
Inositol Signaling Group, National Institute of Environmental Health Sciences, National Institutes of Health, U.S. Department of Health and Human Services, P.O. Box 12233, Research Triangle Park, North Carolina 27709, USA.
Wang
Xueqing
X
Zhang
Guangping
G
Gentzsch
Martina
M
Nelson
Deborah J
DJ
Shears
Stephen B
SB
D000818:Animals; D018938:Caco-2 Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D006224:Cricetinae; D004594:Electrophysiology; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D015398:Signal Transduction
D018118:Chloride Channels; D007295:Inositol Phosphates; C081652:inositol-3,4,5,6-tetrakisphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2